On cherche à étudier l’effet de trois facteurs sur le transcriptome des racines d’Arabidopsis thaliana. Le CO2, au cours des études préliminaires, s’est montré peu influent en conditions contrôles de fer et de nitrates, et accentué en cas de stress nutritionnel. Nous reprennons ces résultats avec des fonctions génériques et propres pour en faire le résumé et de jolis graphes.

## Warning in scan(file = file, what = what, sep = sep, quote = quote, dec = dec, :
## EOF within quoted string
## Warning in scan(file = file, what = what, sep = sep, quote = quote, dec = dec, :
## number of items read is not a multiple of the number of columns

Import des données : matrice d’expression

On a, pour chaque gène et chaque condition, son niveau d’expression en sortie de quantification. On labelle les conditions avec le code suivant : lettre majuscule pour le niveau fort, minuscule pour le niveau faible. Le réplicat est donné après l’underscore.

[1] "cnF_3"
[1] At_AmbientCO2_LowNitrate_Fe1
48 Levels: At_AmbientCO2_HighNitrate_Fe1 ... Sl_ElevatedCO2_LowNitrate_FeStarvation3
[1] "At_AmbientCO2_LowNitrate_Fe"
          cNF_3 cNF_2 cNF_1 cnF_2 cnF_1 cnF_3 CNF_1 CnF_2 CnF_1 CnF_3 cNf_1
AT1G01010  1526  1006  1116  1275   967  1018   854  1132  1294  1364  2325
AT1G01020   416   285   289   349   364   370   226   513   502   561   461
AT1G01030    31    15    19    29    36    28    12    47    34    47    18
          cnf_2 cnf_1 cNf_2 cNf_3 cnf_3 Cnf_3 CNf_1 Cnf_1 Cnf_2 CNf_3 CNf_2
AT1G01010  2113  2193  2564  2364  2074  1987  2027  1754  1697  1537  1898
AT1G01020   407   432   614   380   502   484   467   426   415   413   462
AT1G01030    40    32    44    37    27    42    39    36    40    37    37
          CNF_3 CNF_2
AT1G01010   816   912
AT1G01020   223   312
AT1G01030    15    19
 [ reached 'max' / getOption("max.print") -- omitted 3 rows ]
[1] 23342    24

Effet CO2 en conditions contrôle

On définit les conditions contrôle comme suit : fort nitrate et fort fer.

  (Intercept) groupCNF
1           1        0
2           1        0
3           1        0
4           1        1
5           1        1
6           1        1
attr(,"assign")
[1] 0 1
attr(,"contrasts")
attr(,"contrasts")$group
[1] "contr.treatment"

[1] "cNF_3" "cNF_2" "cNF_1" "CNF_1" "CNF_3" "CNF_2"
    cNF_3     cNF_2     cNF_1     CNF_1     CNF_3     CNF_2 
1.0090325 1.0047763 0.9805842 1.0116521 0.9699686 1.0239863 

[1] "68  genes DE"

[1] 17

Low nitrate

  (Intercept) groupCnF
1           1        0
2           1        0
3           1        0
4           1        1
5           1        1
6           1        1
attr(,"assign")
[1] 0 1
attr(,"contrasts")
attr(,"contrasts")$group
[1] "contr.treatment"

[1] "cnF_2" "cnF_1" "cnF_3" "CnF_2" "CnF_1" "CnF_3"
    cnF_2     cnF_1     cnF_3     CnF_2     CnF_1     CnF_3 
0.9862253 0.9873030 0.9924054 1.0167225 1.0047221 1.0126218 

[1] "1312  genes DE"

Low iron

  (Intercept) groupCNf
1           1        0
2           1        0
3           1        0
4           1        1
5           1        1
6           1        1
attr(,"assign")
[1] 0 1
attr(,"contrasts")
attr(,"contrasts")$group
[1] "contr.treatment"

[1] "cNf_1" "cNf_2" "cNf_3" "CNf_1" "CNf_3" "CNf_2"
    cNf_1     cNf_2     cNf_3     CNf_1     CNf_3     CNf_2 
0.9799002 1.0044883 0.9723876 0.9981139 1.0241404 1.0209697 

[1] "2219  genes DE"

Low iron and nitrate

  (Intercept) groupCnf
1           1        0
2           1        0
3           1        0
4           1        1
5           1        1
6           1        1
attr(,"assign")
[1] 0 1
attr(,"contrasts")
attr(,"contrasts")$group
[1] "contr.treatment"

[1] "cnf_2" "cnf_1" "cnf_3" "Cnf_3" "Cnf_1" "Cnf_2"
    cnf_2     cnf_1     cnf_3     Cnf_3     Cnf_1     Cnf_2 
0.9975633 1.0046643 1.0015093 0.9727479 1.0064673 1.0170479 

[1] "1656  genes DE"

Venn diagram

On visualise les gènes différentiellement exprimés en commun entre les différents niveaux des autres facteurs.

    gene_id   a.value    m.value      p.value      q.value rank estimatedDEG
1 AT5G64120  8.797218 -1.1121447 1.895561e-15 4.424619e-11    1            1
2 AT1G19250  9.948535 -0.8706103 1.978672e-14 1.946612e-10    2            1
3 AT4G25100  9.324940 -0.9038503 2.501858e-14 1.946612e-10    3            1
4 AT5G59520  8.819711 -1.2066060 1.693286e-12 9.037014e-09    4            1
5 AT2G01520 14.038784 -0.9716064 1.935784e-12 9.037014e-09    5            1
6 AT1G67270  5.104540 -2.2140173 3.326926e-12 1.294285e-08    6            1
7 AT5G23980  8.719121 -1.1156310 2.014845e-10 6.718644e-07    7            1
8 AT4G37060 10.229806 -0.8191550 6.994317e-10 2.040767e-06    8            1
9 AT5G23990  4.823373 -2.0335514 9.553935e-10 2.477866e-06    9            1
  upreg
1     0
2     0
3     0
4     0
5     0
6     0
7     0
8     0
9     0
 [ reached 'max' / getOption("max.print") -- omitted 59 rows ]
    gene_id   a.value   m.value      p.value      q.value rank estimatedDEG
1 AT5G61600 10.978022  1.715345 5.768501e-56 1.346484e-51    1            1
2 AT4G27450 10.987889  1.361106 1.241560e-51 1.449024e-47    2            1
3 AT1G27730  9.421760  2.187544 2.462590e-51 1.916059e-47    3            1
4 AT5G51190  8.417337  2.782680 4.320997e-49 2.521518e-45    4            1
5 AT2G27830  9.879502  1.316552 1.397834e-39 6.525650e-36    5            1
6 AT1G19530 12.542251  1.538979 1.842265e-38 7.167027e-35    6            1
7 AT4G27280 10.488397  1.691216 4.608884e-38 1.536865e-34    7            1
8 AT5G13930 10.407278 -1.747350 6.719526e-38 1.960590e-34    8            1
9 AT3G44260  8.737846  2.483672 1.820216e-36 4.720832e-33    9            1
  upreg
1     1
2     1
3     1
4     1
5     1
6     1
7     1
8     0
9     1
 [ reached 'max' / getOption("max.print") -- omitted 1303 rows ]
    gene_id   a.value   m.value       p.value       q.value rank estimatedDEG
1 AT1G78340 10.002535 -3.261719 5.399049e-136 1.260246e-131    1            1
2 AT4G13180 10.301728 -2.811270 3.011796e-121 3.515067e-117    2            1
3 AT4G15550  8.217350 -3.781600 5.522983e-117 4.297249e-113    3            1
4 AT4G34131  8.946763 -2.699004  1.584031e-95  9.243612e-92    4            1
5 AT1G76680  9.778808 -3.051667  2.789158e-90  1.302091e-86    5            1
6 AT3G28345  9.654586 -2.538451  3.170729e-89  1.233519e-85    6            1
7 AT3G45060  9.161753  2.510301  5.415418e-82  1.805810e-78    7            1
8 AT1G76690  9.762616 -2.235867  5.461401e-76  1.593500e-72    8            1
9 AT3G28740  4.011621 -7.093650  9.075419e-76  2.353760e-72    9            1
  upreg
1     0
2     0
3     0
4     0
5     0
6     0
7     1
8     0
9     0
 [ reached 'max' / getOption("max.print") -- omitted 2210 rows ]
    gene_id   a.value   m.value      p.value      q.value rank estimatedDEG
1 AT3G49620  8.971142  3.325629 4.576332e-77 1.068207e-72    1            1
2 AT2G26400 12.032573 -2.107176 1.946526e-64 2.271791e-60    2            1
3 AT1G21100  9.986600  1.996729 3.562033e-43 2.771499e-39    3            1
4 AT1G69530  9.527594 -1.507353 2.066807e-40 1.206085e-36    4            1
5 AT5G51860  6.042441  3.573607 4.906245e-40 2.290431e-36    5            1
6 AT5G51870  8.032896  1.826646 6.948595e-39 2.703235e-35    6            1
7 AT1G08630  8.692334  2.049414 1.918515e-37 6.397426e-34    7            1
8 AT1G56600  8.952644 -1.850385 1.108793e-36 3.235181e-33    8            1
9 AT1G43800 11.887403 -2.934754 2.086789e-36 5.412203e-33    9            1
  upreg
1     1
2     0
3     1
4     0
5     1
6     1
7     1
8     0
9     0
 [ reached 'max' / getOption("max.print") -- omitted 1647 rows ]

   ensembl_gene_id
1        AT1G02640
2        AT1G11185
3        AT1G11260
4        AT1G11920
5        AT1G12040
6        AT1G12805
7        AT1G17180
8        AT1G18400
9        AT1G19250
10       AT1G27950
11       AT1G29100
12       AT1G33055
13       AT1G43800
14       AT1G48930
15       AT1G54950
16       AT1G54970
17       AT1G55790
18       AT1G56580
                                                                                                                                                                                                                                            description
1                                                                                                                                                                                 Probable beta-D-xylosidase 2 [Source:UniProtKB/Swiss-Prot;Acc:Q94KD8]
2                                                                                                                                                                                                                 other RNA [Source:TAIR;Acc:AT1G11185]
3                                                                                                                                                                                                         STP1 [Source:UniProtKB/TrEMBL;Acc:A0A178WJ63]
4                                                                                                                                                                                     Putative pectate lyase 2 [Source:UniProtKB/Swiss-Prot;Acc:O65388]
5                                                                                                                                                                  Leucine-rich repeat extensin-like protein 1 [Source:UniProtKB/Swiss-Prot;Acc:O65375]
6                                                                                                                                                                                       Nucleotide binding protein [Source:UniProtKB/TrEMBL;Acc:Q3EDD5]
7                                                                                                                                                                                Glutathione S-transferase U25 [Source:UniProtKB/Swiss-Prot;Acc:Q9SHH7]
8                                                                                                                                                                                   Transcription factor BEE 1 [Source:UniProtKB/Swiss-Prot;Acc:Q8GZ13]
9                                                                                                                                                                   Probable flavin-containing monooxygenase 1 [Source:UniProtKB/Swiss-Prot;Acc:Q9LMA1]
10                                                                                                                                                          Non-specific lipid transfer protein GPI-anchored 1 [Source:UniProtKB/Swiss-Prot;Acc:Q9C7F7]
11                                                                                                                                                                 Heavy metal transport/detoxification superfamily protein [Source:TAIR;Acc:AT1G29100]
12                                                                                                                                              Uncharacterized protein unannotated coding sequence from BAC F9L11 [Source:UniProtKB/TrEMBL;Acc:Q94K21]
13                                                                                                                                               Stearoyl-[acyl-carrier-protein] 9-desaturase 6, chloroplastic [Source:UniProtKB/Swiss-Prot;Acc:Q84VY3]
14                                                                                                                                                                                             Endoglucanase 5 [Source:UniProtKB/Swiss-Prot;Acc:Q9M995]
15 unknown protein; FUNCTIONS IN: molecular_function unknown; INVOLVED IN: biological_process unknown; LOCATED IN: endomembrane system; BEST Arabidopsis thaliana protein match is: unknown protein (TAIR:AT1G07690.1); Ha. [Source:TAIR;Acc:AT1G54950]
16                                                                                                                                                                                      Proline-rich protein 1 [Source:UniProtKB/Swiss-Prot;Acc:Q9FZ35]
17                                                                                                                                                                                     Domain of unknown function (DUF2431) [Source:TAIR;Acc:AT1G55790]
18                                                                                                                                                                                             At1g56580/F25P12_18 [Source:UniProtKB/TrEMBL;Acc:Q9FXB0]
   external_gene_name entrezgene_id
1                BXL2        837940
2                                NA
3                STP1        837667
4                            837742
5                LRX1        837756
6                           2745749
7              GSTU25        838289
8                BEE1        838421
9                FMO1        838508
10              LTPG1        839688
11                           839785
12                           840201
13         S-ACP-DES6        840977
14            AtGH9C1        841315
15                           841935
16               PRP1        841938
17                               NA
18                SVB        842112
 [ reached 'max' / getOption("max.print") -- omitted 82 rows ]

Investigations

Double carence en CO2 ambient et élevé

On s’interroge sur l’effet de la double carence fer et nitrate en CO2 ambiant et élevé. On ne l’avait pas fait car cela fait varer deux facteurs à la fois.

  (Intercept) groupcNF
1           1        0
2           1        0
3           1        0
4           1        1
5           1        1
6           1        1
attr(,"assign")
[1] 0 1
attr(,"contrasts")
attr(,"contrasts")$group
[1] "contr.treatment"

[1] "cnf_2" "cnf_1" "cnf_3" "cNF_3" "cNF_2" "cNF_1"
    cnf_2     cnf_1     cnf_3     cNF_3     cNF_2     cNF_1 
1.0242524 1.0393358 1.0253221 0.9831632 0.9914864 0.9364401 

[1] "9069  genes DE"

  (Intercept) groupCNF
1           1        0
2           1        0
3           1        0
4           1        1
5           1        1
6           1        1
attr(,"assign")
[1] 0 1
attr(,"contrasts")
attr(,"contrasts")$group
[1] "contr.treatment"

[1] "Cnf_3" "Cnf_1" "Cnf_2" "CNF_1" "CNF_3" "CNF_2"
    Cnf_3     Cnf_1     Cnf_2     CNF_1     CNF_3     CNF_2 
0.9823882 1.0318759 1.0281336 1.0008911 0.9447685 1.0119428 

[1] "8309  genes DE"

   ensembl_gene_id
1        AT1G01720
2        AT1G01750
3        AT1G02575
4        AT1G02640
5        AT1G02850
6        AT1G03790
7        AT1G03870
8        AT1G04250
9        AT1G05650
10       AT1G05660
11       AT1G06120
12       AT1G07680
13       AT1G07690
14       AT1G07750
15       AT1G08080
16       AT1G08100
17       AT1G08290
18       AT1G08500
                                                                                                                                                                                                                                                                                                                                   description
1                                                                                                                                                                                                                                                                     NAC domain-containing protein 2 [Source:UniProtKB/Swiss-Prot;Acc:Q39013]
2                                                                                                                                                                                                                                                                                   actin depolymerizing factor 11 [Source:TAIR;Acc:AT1G01750]
3                                                                                                                                                                                                                 unknown protein; BEST Arabidopsis thaliana protein match is: unknown protein (TAIR:AT1G02570.1). [Source:TAIR;Acc:AT1G02575]
4                                                                                                                                                                                                                                                                        Probable beta-D-xylosidase 2 [Source:UniProtKB/Swiss-Prot;Acc:Q94KD8]
5                                                                                                                                                                                                                                                                                 Beta-glucosidase 11 [Source:UniProtKB/Swiss-Prot;Acc:B3H5Q1]
6                                                                                                                                                                                                                                                        Zinc finger CCCH domain-containing protein 2 [Source:UniProtKB/Swiss-Prot;Acc:Q9ZWA1]
7                                                                                                                                                                                                                                                                                               At1g03870 [Source:UniProtKB/TrEMBL;Acc:B3LF88]
8                                                                                                                                                                                                                                                                      Auxin-responsive protein IAA17 [Source:UniProtKB/Swiss-Prot;Acc:P93830]
9                                                                                                                                                                                                                                                                                        F3F20.10 protein [Source:UniProtKB/TrEMBL;Acc:Q9SYK6]
10                                                                                                                                                                                                                                                                                       F3F20.11 protein [Source:UniProtKB/TrEMBL;Acc:Q9SYK7]
11                                                                                                                                                                                                                                                                  Delta-9 desaturase-like 3 protein [Source:UniProtKB/Swiss-Prot;Acc:Q9FPD5]
12                                                                                                                                                                                                                                                                                               F24B9.23 [Source:UniProtKB/TrEMBL;Acc:Q9LQP3]
13 FUNCTIONS IN: molecular_function unknown; INVOLVED IN: biological_process unknown; LOCATED IN: endomembrane system; EXPRESSED IN: root; CONTAINS InterPro DOMAIN/s: Orthoreovirus membrane fusion p10 (InterPro:IPR009854); BEST Arabidopsis thaliana pro /.../atch is: unknown protein (TAIR:AT1G54950.1); Ha. [Source:TAIR;Acc:AT1G07690]
14                                                                                                                                                                                                                                                                                     At1g07750/F24B9_13 [Source:UniProtKB/TrEMBL;Acc:Q9LQQ3]
15                                                                                                                                                                                                                                                                         Alpha carbonic anhydrase 7 [Source:UniProtKB/Swiss-Prot;Acc:Q8L817]
16                                                                                                                                                                                                                                                                                               NRT2 [Source:UniProtKB/TrEMBL;Acc:A0A178W7F7]
17                                                                                                                                                                                                                                                                           Zinc finger protein WIP3 [Source:UniProtKB/Swiss-Prot;Acc:Q9SGD1]
18                                                                                                                                                                                                                                                                          Early nodulin-like protein 18 [Source:UniProtKB/TrEMBL;Acc:O82083]
   external_gene_name entrezgene_id
1              NAC002        839265
2               ADF11        839281
3                          10723100
4                BXL2        837940
5              BGLU11        839435
6                 SOM        839408
7                FLA9        839384
8               IAA17        839568
9                            837072
10                           837073
11                           837121
12                           837281
13                           837282
14                           837290
15               ACA7        837326
16             NRT2.2        837328
17               WIP3        837349
18            ENODL18        837371
 [ reached 'max' / getOption("max.print") -- omitted 6632 rows ]

On trouve énormément de gènes, ce qui est cohérent car le nitrate et le fer pris séparément avaient déjà beaucoup d’effet.

DEG cNF et CNF avec un autre condition

On cherche à savoir si il n’y pas pas un soucis avec l’échantillon CNF, qui semble avoi très très peu de différences avec cNF. Aurait-on envoyé les mêmes tubes?

On ne voit que très peu de DEGs en comparant directement cNF et CNF, on cherche donc à savoir si on trouve le même nombre de DEGs quand on compare cNF - x et CNF -x, ce qui validerait l’hypothèse d’un transcriptôme quasi identique. On choisit ici par exemple \(x=\) cnF.

Il semble qu’il y ait quand même pas mal de différences entre ces transcriptomes, car quand on les compare au même troisième transcriptome on a une différence de 1000 gènes (1900 contre 2800). Le diagramme de Venn montre que ces gènes sont en partie différents. Les transcriptômes ont bien l’air différents. Si on prend un autre x :

On va faire les corrélations d’expression entre ces deux conditions.

[1] 0.9938199 0.9906937 0.9810858 0.9860430 0.9921428 0.9860068

cNF et CNF semblent bien plus proches entre eux que cNF ne l’est avec tous les autres.

 

A work by Océane Cassan

oceane.cassan@supagro.fr